White blister rust, caused by the oomycete Albugo candida, is a widespread disease of Brassica cr... more White blister rust, caused by the oomycete Albugo candida, is a widespread disease of Brassica crops. The Arabidopsis CSA1/DAR4 (also known as CSA1/CHS3) paired immune receptor carries an Integrated Domain (ID) with homology to the DA1 family of peptidases. Using domain swaps with DA1 family members, we show that the DAR4 ID acts as an integrated decoy for DAR3, which interacts with and inhibits the peptidase activities of DA1, DAR1 and DAR2 family members. Albugo infection rapidly lowered DAR3 levels and activates DA1 peptidase activity. This promotes endoreduplication of host tissues to support pathogen growth. We propose that DAR4/CSA1 senses the actions of a putative Albugo effector that reduces DAR3 levels and initiates defense.
Plant disease resistance involves both detection of microbial molecular patterns by cell-surface ... more Plant disease resistance involves both detection of microbial molecular patterns by cell-surface pattern recognition receptors and detection of pathogen effectors by intracellular NLR immune receptors. NLRs are classified as sensor NLRs, involved in effector detection, or helper NLRs required for sensor NLR signalling. TIR-domain-containing sensor NLRs (TNLs) require helper NLRs NRG1 and ADR1 for resistance, and their activation of defense also requires the lipase-domain proteins EDS1, SAG101 and PAD4. We investigated how the helper NLR NRG1 supports TNL-initiated immunity with EDS1 and SAG101. We find that NRG1 associates with EDS1 and SAG101 at the plasma membrane and in the nucleus, but only self-associates at the plasma membrane. Activation of TNLs is sufficient to trigger NRG1-EDS1-SAG101 interaction, but cell-surface receptor-initiated defense is also required to form an oligomeric Resistosome. The data point to formation of NRG1-EDS1-SAG101 heterotrimers in the nucleus upon i...
Late blight caused by the oomycete pathogenPhytophthora infestanscontinues to cause major worldwi... more Late blight caused by the oomycete pathogenPhytophthora infestanscontinues to cause major worldwide losses in potato and tomato. Most accessions ofSolanum americanum, a globally distributed, wild Solanaceae plant, are highly resistant to late blight. We generated high-quality reference genomes of fourS. americanumaccessions, re-sequenced 52 accessions, and we defined variation in the NLR immune receptor genes (theS. americanumNLRome). We further screened for variation in recognition of ∼315P. infestansRXLR effectors in 52S. americanumaccessions. Using these genotypic and phenotypic data, we cloned three novel NLR-encoding genesRpi-amr4, Rpi-amr16andRpi-amr17, and determined their corresponding RXLR effector genesAvramr4(PITG_22825),Avramr16(PITG_02860) andAvramr17(PITG_04373) fromP. infestans. These genomic resources and methodology will support efforts to convert potato into a “nonhost” of late blight and can be applied to diseases of other crops.
Progeny of tobacco line 2853.6, which carries a streptomycin phosphotransferase (SPT) gene interr... more Progeny of tobacco line 2853.6, which carries a streptomycin phosphotransferase (SPT) gene interrupted by the maize element Activator (Ac), were selected for streptomycin resistance (Spr) because of germinal Ac excision. Some events gave rise to Spr alleles that were unstable and exhibited a mottled phenotype on streptomycin-containing medium due to somatic loss of SPT function. This instability was most pronounced in one particular line, Spr12F. Other Spr alleles rarely exhibited silencing of SPT. Streptomycin-sensitive, homozygous Spr12F plants were recovered, and crosses were performed with other, more stable Spr lines. A high proportion of the resulting heterozygous progeny were silenced for SPT expression. The silenced state was heritable even after the Spr12F allele segregated away. No correlation could be made between silencing and methylation of the SPT gene. Structural analysis of allele Spr12F showed that the SPT gene from which Ac had excised was flanked by direct repeats...
Proceedings of the National Academy of Sciences of the United States of America, Jan 2, 2012
Gram-negative phytopathogenic bacteria translocate effector proteins into plant cells to subvert ... more Gram-negative phytopathogenic bacteria translocate effector proteins into plant cells to subvert host defenses. These effectors can be recognized by plant nucleotide-binding-leucine-rich repeat immune receptors, triggering defense responses that restrict pathogen growth. AvrRps4, an effector protein from Pseudomonas syringae pv. pisi, triggers RPS4-dependent immunity in resistant accessions of Arabidopsis. To better understand the molecular basis of AvrRps4-triggered immunity, we determined the crystal structure of processed AvrRps4 (AvrRps4(C), residues 134-221), revealing that it forms an antiparallel α-helical coiled coil. Structure-informed mutagenesis reveals an electronegative surface patch in AvrRps4(C) required for recognition by RPS4; mutations in this region can also uncouple triggering of the hypersensitive response from disease resistance. This uncoupling may result from a lower level of defense activation, sufficient for avirulence but not for triggering a hypersensitiv...
SummaryGenome sequences of plant fungal pathogens have enabled the identification of effectors th... more SummaryGenome sequences of plant fungal pathogens have enabled the identification of effectors that cooperatively modulate the cellular environment for successful fungal growth and suppress host defense. Identification and characterization of novel effector proteins are crucial for understanding pathogen virulence and host‐plant defense mechanisms. Previous reports indicate that the Pseudomonas syringae pv. tomato DC3000 type III secretion system (T3SS) can be used to study how non‐bacterial effectors manipulate dicot plant cell function using the effector detector vector (pEDV) system. Here we report a pEDV‐based effector delivery system in which the T3SS of Burkholderia glumae, an emerging rice pathogen, is used to translocate the AVR‐Pik and AVR‐Pii effectors of the fungal pathogen Magnaporthe oryzae to rice cytoplasm. The translocated AVR‐Pik and AVR‐Pii showed avirulence activity when tested in rice cultivars containing the cognate R genes. AVR‐Pik reduced and delayed the hyper...
Plants have evolved a powerful immune system to defend against infection by most microbial organi... more Plants have evolved a powerful immune system to defend against infection by most microbial organisms. However, successful pathogens, such as Pseudomonas syringae , have developed countermeasures and inject virulence proteins into the host plant cell to suppress immunity and cause devastating diseases. Despite intensive research efforts, the molecular targets of bacterial virulence proteins that are important for plant disease development have remained obscure. Here, we show that a conserved P. syringae virulence protein, HopM1, targets an immunity-associated protein, AtMIN7, in Arabidopsis thaliana . HopM1 mediates the destruction of AtMIN7 via the host proteasome. Our results illustrate a strategy by which a bacterial pathogen exploits the host proteasome to subvert host immunity and causes infection in plants.
Proceedings of the National Academy of Sciences, 1999
A major class of plant disease resistance ( R ) genes encodes leucine-rich-repeat proteins that p... more A major class of plant disease resistance ( R ) genes encodes leucine-rich-repeat proteins that possess a nucleotide binding site and amino-terminal similarity to the cytoplasmic domains of the Drosophila Toll and human IL-1 receptors. In Arabidopsis thaliana , EDS1 is indispensable for the function of these R genes. The EDS1 gene was cloned by targeted transposon tagging and found to encode a protein that has similarity in its amino-terminal portion to the catalytic site of eukaryotic lipases. Thus, hydrolase activity, possibly on a lipid-based substrate, is anticipated to be central to EDS1 function. The predicted EDS1 carboxyl terminus has no significant sequence homologies, although analysis of eight defective eds1 alleles reveals it to be essential for EDS1 function. Two plant defense pathways have been defined previously that depend on salicylic acid, a phenolic compound, or jasmonic acid, a lipid-derived molecule. We examined the expression of EDS1 mRNA and marker mRNAs ( PR1...
Proceedings of the National Academy of Sciences, 2001
Reactive oxygen intermediates (ROI) are strongly associated with plant defense responses. The ori... more Reactive oxygen intermediates (ROI) are strongly associated with plant defense responses. The origin of these ROI has been controversial. Arabidopsis respiratory burst oxidase homologues ( rboh genes) have been proposed to play a role in ROI generation. We analyzed lines carrying dSpm insertions in the highly expressed AtrbohD and AtrbohF genes. Both are required for full ROI production observed during incompatible interactions with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000( avrRpm1 ) and the oomycete parasite Peronospora parasitica . We also observed reduced cell death, visualized by trypan blue stain and reduced electrolyte leakage, in the Atrboh mutants after DC3000( avrRpm1 ) inoculation. However, enhanced cell death is observed after infection of mutant lines with P. parasitica . Paradoxically, although atrbohD mutation eliminated the majority of total ROI production, atrbohF mutation exhibited the strongest effect on cell death.
Late blight, caused by the notorious pathogen Phytophthora infestans, is a devastating disease of... more Late blight, caused by the notorious pathogen Phytophthora infestans, is a devastating disease of potato (Solanum tuberosum) and tomato (Solanum lycopersicum), and during the 1840s caused the Irish potato famine and over one million fatalities. Currently, grown potato cultivars lack adequate blight tolerance. Earlier cultivars bred for resistance used disease resistance genes that confer immunity only to some strains of the pathogen harboring corresponding avirulence gene. Specific resistance gene-mediated immunity and chemical controls are rapidly overcome in the field when new pathogen races arise through mutation, recombination, or migration from elsewhere. A mitogen-activated protein kinase (MAPK) cascade plays a pivotal role in plant innate immunity. Here we show that the transgenic potato plants that carry a constitutively active form of MAPK kinase driven by a pathogen-inducible promoter of potato showed high resistance to early blight pathogen Alternaria solani as well as P....
White blister rust, caused by the oomycete Albugo candida, is a widespread disease of Brassica cr... more White blister rust, caused by the oomycete Albugo candida, is a widespread disease of Brassica crops. The Arabidopsis CSA1/DAR4 (also known as CSA1/CHS3) paired immune receptor carries an Integrated Domain (ID) with homology to the DA1 family of peptidases. Using domain swaps with DA1 family members, we show that the DAR4 ID acts as an integrated decoy for DAR3, which interacts with and inhibits the peptidase activities of DA1, DAR1 and DAR2 family members. Albugo infection rapidly lowered DAR3 levels and activates DA1 peptidase activity. This promotes endoreduplication of host tissues to support pathogen growth. We propose that DAR4/CSA1 senses the actions of a putative Albugo effector that reduces DAR3 levels and initiates defense.
Plant disease resistance involves both detection of microbial molecular patterns by cell-surface ... more Plant disease resistance involves both detection of microbial molecular patterns by cell-surface pattern recognition receptors and detection of pathogen effectors by intracellular NLR immune receptors. NLRs are classified as sensor NLRs, involved in effector detection, or helper NLRs required for sensor NLR signalling. TIR-domain-containing sensor NLRs (TNLs) require helper NLRs NRG1 and ADR1 for resistance, and their activation of defense also requires the lipase-domain proteins EDS1, SAG101 and PAD4. We investigated how the helper NLR NRG1 supports TNL-initiated immunity with EDS1 and SAG101. We find that NRG1 associates with EDS1 and SAG101 at the plasma membrane and in the nucleus, but only self-associates at the plasma membrane. Activation of TNLs is sufficient to trigger NRG1-EDS1-SAG101 interaction, but cell-surface receptor-initiated defense is also required to form an oligomeric Resistosome. The data point to formation of NRG1-EDS1-SAG101 heterotrimers in the nucleus upon i...
Late blight caused by the oomycete pathogenPhytophthora infestanscontinues to cause major worldwi... more Late blight caused by the oomycete pathogenPhytophthora infestanscontinues to cause major worldwide losses in potato and tomato. Most accessions ofSolanum americanum, a globally distributed, wild Solanaceae plant, are highly resistant to late blight. We generated high-quality reference genomes of fourS. americanumaccessions, re-sequenced 52 accessions, and we defined variation in the NLR immune receptor genes (theS. americanumNLRome). We further screened for variation in recognition of ∼315P. infestansRXLR effectors in 52S. americanumaccessions. Using these genotypic and phenotypic data, we cloned three novel NLR-encoding genesRpi-amr4, Rpi-amr16andRpi-amr17, and determined their corresponding RXLR effector genesAvramr4(PITG_22825),Avramr16(PITG_02860) andAvramr17(PITG_04373) fromP. infestans. These genomic resources and methodology will support efforts to convert potato into a “nonhost” of late blight and can be applied to diseases of other crops.
Progeny of tobacco line 2853.6, which carries a streptomycin phosphotransferase (SPT) gene interr... more Progeny of tobacco line 2853.6, which carries a streptomycin phosphotransferase (SPT) gene interrupted by the maize element Activator (Ac), were selected for streptomycin resistance (Spr) because of germinal Ac excision. Some events gave rise to Spr alleles that were unstable and exhibited a mottled phenotype on streptomycin-containing medium due to somatic loss of SPT function. This instability was most pronounced in one particular line, Spr12F. Other Spr alleles rarely exhibited silencing of SPT. Streptomycin-sensitive, homozygous Spr12F plants were recovered, and crosses were performed with other, more stable Spr lines. A high proportion of the resulting heterozygous progeny were silenced for SPT expression. The silenced state was heritable even after the Spr12F allele segregated away. No correlation could be made between silencing and methylation of the SPT gene. Structural analysis of allele Spr12F showed that the SPT gene from which Ac had excised was flanked by direct repeats...
Proceedings of the National Academy of Sciences of the United States of America, Jan 2, 2012
Gram-negative phytopathogenic bacteria translocate effector proteins into plant cells to subvert ... more Gram-negative phytopathogenic bacteria translocate effector proteins into plant cells to subvert host defenses. These effectors can be recognized by plant nucleotide-binding-leucine-rich repeat immune receptors, triggering defense responses that restrict pathogen growth. AvrRps4, an effector protein from Pseudomonas syringae pv. pisi, triggers RPS4-dependent immunity in resistant accessions of Arabidopsis. To better understand the molecular basis of AvrRps4-triggered immunity, we determined the crystal structure of processed AvrRps4 (AvrRps4(C), residues 134-221), revealing that it forms an antiparallel α-helical coiled coil. Structure-informed mutagenesis reveals an electronegative surface patch in AvrRps4(C) required for recognition by RPS4; mutations in this region can also uncouple triggering of the hypersensitive response from disease resistance. This uncoupling may result from a lower level of defense activation, sufficient for avirulence but not for triggering a hypersensitiv...
SummaryGenome sequences of plant fungal pathogens have enabled the identification of effectors th... more SummaryGenome sequences of plant fungal pathogens have enabled the identification of effectors that cooperatively modulate the cellular environment for successful fungal growth and suppress host defense. Identification and characterization of novel effector proteins are crucial for understanding pathogen virulence and host‐plant defense mechanisms. Previous reports indicate that the Pseudomonas syringae pv. tomato DC3000 type III secretion system (T3SS) can be used to study how non‐bacterial effectors manipulate dicot plant cell function using the effector detector vector (pEDV) system. Here we report a pEDV‐based effector delivery system in which the T3SS of Burkholderia glumae, an emerging rice pathogen, is used to translocate the AVR‐Pik and AVR‐Pii effectors of the fungal pathogen Magnaporthe oryzae to rice cytoplasm. The translocated AVR‐Pik and AVR‐Pii showed avirulence activity when tested in rice cultivars containing the cognate R genes. AVR‐Pik reduced and delayed the hyper...
Plants have evolved a powerful immune system to defend against infection by most microbial organi... more Plants have evolved a powerful immune system to defend against infection by most microbial organisms. However, successful pathogens, such as Pseudomonas syringae , have developed countermeasures and inject virulence proteins into the host plant cell to suppress immunity and cause devastating diseases. Despite intensive research efforts, the molecular targets of bacterial virulence proteins that are important for plant disease development have remained obscure. Here, we show that a conserved P. syringae virulence protein, HopM1, targets an immunity-associated protein, AtMIN7, in Arabidopsis thaliana . HopM1 mediates the destruction of AtMIN7 via the host proteasome. Our results illustrate a strategy by which a bacterial pathogen exploits the host proteasome to subvert host immunity and causes infection in plants.
Proceedings of the National Academy of Sciences, 1999
A major class of plant disease resistance ( R ) genes encodes leucine-rich-repeat proteins that p... more A major class of plant disease resistance ( R ) genes encodes leucine-rich-repeat proteins that possess a nucleotide binding site and amino-terminal similarity to the cytoplasmic domains of the Drosophila Toll and human IL-1 receptors. In Arabidopsis thaliana , EDS1 is indispensable for the function of these R genes. The EDS1 gene was cloned by targeted transposon tagging and found to encode a protein that has similarity in its amino-terminal portion to the catalytic site of eukaryotic lipases. Thus, hydrolase activity, possibly on a lipid-based substrate, is anticipated to be central to EDS1 function. The predicted EDS1 carboxyl terminus has no significant sequence homologies, although analysis of eight defective eds1 alleles reveals it to be essential for EDS1 function. Two plant defense pathways have been defined previously that depend on salicylic acid, a phenolic compound, or jasmonic acid, a lipid-derived molecule. We examined the expression of EDS1 mRNA and marker mRNAs ( PR1...
Proceedings of the National Academy of Sciences, 2001
Reactive oxygen intermediates (ROI) are strongly associated with plant defense responses. The ori... more Reactive oxygen intermediates (ROI) are strongly associated with plant defense responses. The origin of these ROI has been controversial. Arabidopsis respiratory burst oxidase homologues ( rboh genes) have been proposed to play a role in ROI generation. We analyzed lines carrying dSpm insertions in the highly expressed AtrbohD and AtrbohF genes. Both are required for full ROI production observed during incompatible interactions with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000( avrRpm1 ) and the oomycete parasite Peronospora parasitica . We also observed reduced cell death, visualized by trypan blue stain and reduced electrolyte leakage, in the Atrboh mutants after DC3000( avrRpm1 ) inoculation. However, enhanced cell death is observed after infection of mutant lines with P. parasitica . Paradoxically, although atrbohD mutation eliminated the majority of total ROI production, atrbohF mutation exhibited the strongest effect on cell death.
Late blight, caused by the notorious pathogen Phytophthora infestans, is a devastating disease of... more Late blight, caused by the notorious pathogen Phytophthora infestans, is a devastating disease of potato (Solanum tuberosum) and tomato (Solanum lycopersicum), and during the 1840s caused the Irish potato famine and over one million fatalities. Currently, grown potato cultivars lack adequate blight tolerance. Earlier cultivars bred for resistance used disease resistance genes that confer immunity only to some strains of the pathogen harboring corresponding avirulence gene. Specific resistance gene-mediated immunity and chemical controls are rapidly overcome in the field when new pathogen races arise through mutation, recombination, or migration from elsewhere. A mitogen-activated protein kinase (MAPK) cascade plays a pivotal role in plant innate immunity. Here we show that the transgenic potato plants that carry a constitutively active form of MAPK kinase driven by a pathogen-inducible promoter of potato showed high resistance to early blight pathogen Alternaria solani as well as P....
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